Synthesis, characterization of poly l(+) lactic acid and its application in sustained release of isosorbide dinitrate

Poly l(+) lactic acid (PLLA) has become crucial in the biomedical industry for various uses. The direct polycondensation method was used to prepare Poly l(+) Lactic Acid (PLLA). Different catalysts, including metal oxides and metal halides, were used to test the polymerization technique. The effect of the amount of catalysts and the type of coupling agent were investigated. The effect of reaction time and polymerization solvents was also studied. PLLA was loaded with isosorbide dinitrate utilizing the solvent evaporation process. The synthesized polymer-drug system was evaluated by different means such as FT-IR, TGA, DSC, XRD, entrapment efficiency (E.E), drug loading (D.L), particle size analysis, and zeta potential determination. Studies on in-vitro release using UV light at 227 nm at various pH levels were conducted, and the kinetics of release and cytotoxicity using the sulforhodamine B (SRB) assay on human skin fibroblast cells were examined.


FT-IR spectroscopy
Burker's TENSOR 27-series FT-IR measurements in the 400-4000 cm -1 range using KBr pellets to define the functional group structure of samples using chloroform as the solvent.
1 H nuclear and 13 C nuclear magnetic resonance 1 HNMR and 13 CNMR spectra using CDCl 3 on a Bruker Avance II spectrometer running at 400 MHZ were given in ppm to illustrate the chemical configuration and the chemical shifts (δ).

Molecular weight measurements
An Alliance GPCV 2000 (Waters, US) was used to calculate the average molecular weight (Mw) at 30 °C.The mobile phase, tetrahydrofuran (THF), was set at a 1 mL/min flow rate.The polystyrene standards were used to produce the calibration curve.By using gel permeation chromatography (GPC), the Mw and molecular weight distribution index (Mw/Mn) was calculated.The Shodex DEGAS KT-16 de-gasser, the Shodex RI SE-31 RI detector, the Shimadzu C-R7A Chromatopac data processor, and the Shimadzu LC-10A pump made up the analyzer.Chloroform was utilized as the eluent, and two polystyrene gel columns-the Toso TSK gel G4000H8 and G2500H8-were combined.

Viscosity measurement
The measurement of viscosity was conducted by employing Schott Geräte's Cannon-Fenske viscometers, which had a k constant of 0.002061 and a type of 520 00.
At a temperature of 25 °C, the Solomon-Cuita equation was utilized to determine the intrinsic viscosity [η] in chloroform (with a concentration of 2 g/dl) through a single-point evaluation 25 .
where η r = t/t 0 , η sp = η r -1are the relative and the specific viscosity, respectively.c is the concentration of PLLA.

Thermal gravimetric analysis (TGA)
Thermal stability, maximum degradation temperature, and change in mass with temperature were all measured for the samples using TGA analysis.Using a thermal analyzer Perkin Elmer 4000 and a heating rate of 10.0 degrees per minute between 50 and 800 °C.The temperature (T onset ) could be estimated at which thermal degradation started and how the mass changed as the temperature rose.To determine the highest degradation temperature, derived thermogravimetric curves (DrTG) were employed (Tdeg.max .) 28.

Differential scanning calorimetry (DSC)
Polymeric materials are frequently examined using DSC thermograms to identify their thermal transitions.The glass transition temperature (Tg), crystalline temperature (Tc), and melting temperature (Tm) are significant thermal transitions.A Shimadzu DSC60 (Japan) was used to measure the thermal characteristics of the produced polymers at a heating rate of 10 C/min.

X-ray diffraction (XRD)
X-ray pattern was determined using an XMD 300 (Unisantis, Germany).Cu-K radiation, with a wavelength of 1.5406, was run at 45 kV and 0.8 mA with an angle range of 5 to 90, and the scanning rate was set at 0.05/sec.

The particle size & zeta potential measurements
Zetasizer Nano ZS, Malvern, UK, was used to measure the particle.

Scanning electron microscopy (SEM)
The surface morphology of the nanoparticles was observed by SEM analysis using a SU8000 20 kV 4.0 mm X 25.0 K SE(U).Briefly, a thin layer of gold was sputtered onto the particles to facilitate visualization in the SEM.Images were acquired with an electron beam acceleration voltage of 15-20 kV.

Synthesis
Synthesis of PLLA In a round flask fitted with a Dean Starck apparatus and the appropriate amounts of catalyst and coupling agent, 30 mL of xylene and/or a mixture of xylene and decalin were added to 0.1 mol of l-(+) lactic acid 6 .The reaction mixture was agitated at a reflux temperature of 250 °C for 10 to 25 h.Filtering was done on the resultant PLLA.To eliminate the catalyst that hadn't dissolved, PLLA was first dissolved in chloroform.The precipitation of the polymer solution in chloroform was achieved by introducing diethyl ether.A solid or semi-solid product was produced using a rotary evaporator under a vacuum to extract the solvents.The item was cleaned with 100% ethanol before being dried in a vacuum oven at 30 °C.

Effect of solvent types and reaction time
PLLA was polycondensed using two different types of solvents; Xylene alone or in combination with Decalin (1:1) and stannous chloride dihydrate as a catalyst (75 mg).The effect of reaction time was investigated by experimenting twice, once for 10 h and the second for 25 h, with (magnesium oxide 75 mg) as a catalyst.

Effect of catalyst types and their quantity
Magnesium oxide catalyst in varying concentrations was used to study the effects of catalyst type and quantity (50 mg and 75 mg).In addition, different types of catalysts, including magnesium oxide, stannous chloride dihydrate, calcium oxide, aluminum oxide, ferric oxide, zinc chloride, zinc metal, zinc oxide, calcium chloride, antimony trioxide, Arsen III oxide, titanium oxide, strontium chloride, and magnesium metal, were used to investigate the type of catalyst used in the polymerization of PLLA.

Effect of coupling agent types and catalyst/coupling agent ratio in polycondensation of PLLA
To polymerize PLLA, various coupling agents, including p-toluene sulphonic acid, maleic anhydride, and phenyl maleate 29 , were combined with three different catalysts: ZnO, MgO, and ZnCl 2 according to illustrated in Tables 1, 2, 3 and 4.
Table 1.Effect of the ratio between MgO and different coupling in polycondensation of PLLA.

Reaction No. Type of coupling agent
The ratio of MgO/coupling agent 25 mL of acetone was used to dissolve the material in a conical flask with a volume of 250 mL, ranging from 0.2 to 0.5 g.After a five-minute stand-by, phenolphthalein was used as an indicator to titrate the solution with an alcoholic potassium hydroxide solution of 0.1 N. A simultaneous blank analysis without a sample was carried out.Equation (3) denotes the acid value formula that indicates the quantity of potassium hydroxide required to neutralize 1 g of the sample.
N is the KOH solution's normality, W is the sample's weight, and A and B are the amounts of KOH solution consumed by the sample and the blank reading, respectively (g) 27 .

Encapsulation of isosorbide dinitrate in PLLA nanoparticles
Solvent evaporation was the method used in this synthesis.PLLA was dissolved in a 7:1 mixture of dichloromethane and diethyl ether; then, isosorbide dinitrate was added while stirring for an hour to the polymer solution to achieve total miscibility.The surfactant solution in distilled water was combined with the previous drug-containing solution.The mixture was ultrasonically processed for 15 min while being swirled at 700 rpm and kept at 25 °C.After stirring for 15 min, the resultant solution was added to 40 mL of distilled water at 70 °C.When the temperature was raised to (40-45 °C), the solvent vaporized quickly, creating nanoparticles.The mixture was centrifuged at 6000 rpm for 30 min to remove the supernatant water, which was then cooled to room temperature.The remaining nanoparticles were rinsed three times with distilled water before being subjected to lyophilization.

Effect of surfactant type on the nanoparticles size and entrapment efficiency
Several tests were conducted with constant molecular weights of PLLA, constant ratios between polymer and drug, constant surfactant concentrations, and the addition of several types of surfactants to examine the impact of surfactant type on the nanoparticle size and entrapment efficiency.The experiment's conditions are listed in Table 5.The effect of the ratio between PLLA and isosorbide nitrate on the nanoparticles size and entrapment efficiency Several tests were conducted utilizing constant PLLA molecular weights, various polymer and drug ratios, constant surfactant concentrations, and Pluronic F88 as a surfactant to examine the impact of the ratio between PLLA and isosorbide on the nanoparticle size and entrapment efficiency.The experiment's conditions are listed in Table 6.

Effect of molecular weight of PLLA on the nanoparticles size and entrapment efficiency
Various tests employing varied molecular weights of PLLA, a 2:1 ratio between PLLA and drug, constant surfactant concentrations, and Pluronic F88 as a surfactant were conducted to examine the impact of PLLA's molecular weight on the nanoparticle size and entrapment efficiency.The experiment conditions are listed in Table 7.
Using a UV/Vis spectrophotometer to scan an isosorbide dinitrate solution in ethanol, the maximum wavelength detected at 227 nm.To determine entrapment effectiveness, drug loading capacity, and for in vitro release experiments, different concentrations of the drug solution were generated, absorbance was measured, and a calibration curve was created.

In vitro drug release
The behavior of the release of isosorbide dinitrate was investigated using the dialysis technique.In summary, a dialysis membrane with a molecular weight cutoff of 1000 KDa was used.100 mg of the prepared nanoparticles were dissolved in 3 mL of phosphate buffer and placed in a dialysis bag.The bag was immersed in 60 mL of phosphate buffer (pH 7.4, 6.7, 4.5 and 1.2) and gently shaken at regular intervals at 37 °C and 80 rpm.Then two samples of 2 mL each were taken and measured with a UV spectrophotometer at a wavelength of 227 nm.

Cytotoxicity assay
Cell viability was determined by means of the SRB test.After preparing a cell suspension of 5 × 10 -3 cells, 100 μL aliquots were cultured in full medium for a duration of 24 h using 96-well plates.A fresh batch of 100 L of solution with varying amounts of drugs was administered to the cells for treatment.The cells were rendered stable (4) lab.entrapment efficiency = mass of drug encapsulated mass of the initial drug × 100 (5) mass loading capacity = mass of drug encapsulated mass of the polymer × 100  Following the removal of the TCA solution, the cells were rinsed with distilled water on five separate occasions.After placing aliquots of a solution containing SRB in a 70 μL container, the mixture was kept in a dark room at room temperature for 10 min.The dishes were washed thrice using a solution of 1% acetic acid and left to dry in the air for the night.After dissolving the SRB stain that was bound to the protein, 10 mM TRIS was added in 150 μL quantity.The absorbance was measured at 540 nm using a microplate reader called BMG LABTECH ® -FLUOstar Omega (Ortenberg, Germany) 32 .

Results and discussion
According to the procedures outlined in the experimental section, PLLA was synthesized.L-Lactic acid was polycondensed to produce the polymerization utilizing a variety of catalysts.The creation of cyclic by dimer l-Lactic acid is the initial step.The latter was broken down into PLLA by using various coupling agents.The yield ranged from 35 to 90%.The solubility of the drug and polymer was considered when developing the solvent evaporation method to create PLLA/isosorbide dinitrate nanoparticles.

FT-IR spectroscopy
Typical ester absorption peaks were observed in the PLLA FT-IR spectrum at 1761 cm −1 for the -COO-and 1092 cm −1 for the -O-, and at 2948-2998 cm −1 for the -CH 2 -and CH 3 groups.As lactic acid polymerizes, the hydroxyl group bonds with the carboxyl group of another molecule, weakening the significant -OH stretch peak at 3405 cm −1 .The hydroxyl absorption peak almost disappeared in PLA, indicating a decrease in the number of hydroxyl groups, while a larger C = O stretching absorption peak (1761 cm −1 ) was seen during subsequent production steps 33 .Absorption at 870.8 cm −1 indicates the amorphous phase of PLLA, while the crystalline phase is indicated by the band at 755.7 cm −1 (Fig. 1a).Similar outcomes were previously attained using the technique outlined by J. Diani and K. Gall 34 .Isosorbide dinitrate's FT-TR spectra reveal bands (2950-2850 cm −1 ) associated with aliphatic C-H stretching, bands at 1665-1635 cm −1 for asymmetric NO 2 stretching, a band at 1460 cm −1 for methylene scissoring, at 1100 cm −1 due to asymmetric C-O-C stretching, and at 865 cm −1 for O-NO 2 stretching (Fig. 1b).This agrees with the drug's reported process data 35 .
The FT-IR spectra of the encapsulated isosorbide dinitrate showed that the polymer and pure drug maintained their distinct peaks.This indicates that there was no chemical reaction between the drug and polymer during the nanoencapsulation process.Furthermore, the chemical structures of the drug remained unchanged and did www.nature.com/scientificreports/not undergo any degradation.The spectral analysis of PLLA/isosorbide dinitrate nanoparticles did not reveal any significant shift or loss of functional group peaks, indicating the compatibility between PLLA and Isosorbide dinitrate drug (Fig. 1a-c).

HNMR spectra
Various proton resonances were identified in the 1 H NMR spectra, which further elucidate the molecular structure of the synthesized PLLA.Methyl proton resonances were detected at 1.57 ppm, while methane proton resonances appeared at 5.15 ppm within the main chain.In addition, a subtle signal at 4.30 ppm and 1.4 ppm was assigned to the methine proton and the methyl proton near the terminal hydroxyl and carboxyl groups, respectively.The distinct peaks in the spectra confirm the successful preparation of PLLA and are in agreement with similar results obtained by Choubisa et al. 36 who used a solid acid catalyst system for the polycondensation process.The results obtained are in agreement with previous studies where different catalysts were used and where 1 H NMR analysis served as a central tool to characterize the resulting polymers.A comprehensive summary of the spectroscopic data can be found in Table 8 for clarity.

C NMR
The 13 C NMR spectra of PLLA show three distinct peaks at 16.59 ppm, 68.98 ppm and 169.56 ppm, respectively (Table 9).These peaks can be assigned to specific carbon atoms within the PLLA structure, namely the methyl, methane and carbonyl carbons.The resonance positions of these peaks provide valuable insight into the molecular arrangement of PLLA.The observed peaks are in agreement with the results of G. X. Chen et al. who used a similar method for the direct polycondensation of L-lactic acid to synthesize PLLA 37 .This agreement of the results underlines the consistency and reproducibility of the synthesis process and supports the reliability of our PLLA characterization.

Molecular weight analysis and acid value determination
Effect of solvent By employing stannous chloride as a catalyst and xylene as a solvent, PLLA was polymerized to determine the impact of the solvent on the molecular weight of the polymer.Another experiment was run using a 1:1 combination of Xylene and Decaline.For instance, when xylene was used, the PLLA M.wt was 10,099 Dalton, but when xylene and decalin were combined, the PLLA M.wt was 24,874 Dalton.The findings demonstrated that PLLA created using a solvent mixture yielded a greater M.wt than with xylene alone, primarily because Decalin has a higher boiling point than xylene, which facilitates the polycondensation reaction.

Effect of reaction time
By running the reaction for 10 and 25 h with MgO as a catalyst, it was possible to examine the impact of polymerization duration on the M.wt of the resulting PLLA.The response time was lengthened, which enhanced the M.wt of PLLA.

Effect of amount of catalyst
By performing the reaction with 50 mg and 75 mg MgO, it was possible to compare the effects of the two amounts of catalyst on the polymerization of PLLA.According to the data, M.wt produced 8480 Dalton when 75 mg of MgO was used, but M.wt produced 1554 PLLA when 50 mg of MgO was used.The degree of polymerization increased as the catalyst concentration did as well.Table 10 explains this information.

Effect of the ratio between catalyst and coupling agent
The researchers investigated the effects of the relationship between catalyst and coupling agent.In the case of MgO as catalyst and p-toluene sulfonic acid as coupling agent, the results of the experiments showed that a ratio of 2:1 between catalyst and coupling agent gave the best results.This ratio was supported by the high molecular weight produced in this example, which was higher than ratios of 1:1 or 3:1, indicating that a higher catalyst ratio is associated with a higher degree of polymerization.However, the molecular weight of the polymer produced decreased when the catalyst was increased to level 3.The molecular weight increased when maleic anhydride was replaced by p-toluene sulfonic acid at a ratio of 2:1 as a coupling agent (Table 10).
To test the impact of coupling agents on the polymerization process, different coupling agents, such as p-Toluene sulphonic acid, maleic anhydride, and phthalic anhydride, were used with ZnO as a catalyst (Fig. 2).If p-Toluene sulphonic acid is used as the coupling agent and ZnO is used as the catalyst in a 2:1 ratio.This demonstrated the polymer's large molecular weight.The molecular weight of the resulting polymer was enlarged by altering the coupling agent to maleic anhydride.
However, on substituting the coupling agent to Phthalic anhydride with 2:1.The molecular weight did not significantly rise, but it did increase when the ratio of ZnO to phthalic anhydride was changed to 3:1.Maleic anhydride was a successful coupling agent with ZnO in the polymerization of PLLA, according to the data.
In the presence of ZnCl 2 as a catalyst, the impact of coupling agent type was investigated.Based on the outcomes, the molecular weight produced by p-toluene sulphonic acid was higher than that of the other coupling agents.Finally, p-toluene sulphonic acid was a superior coupling agent with metal chloride catalysts than maleic anhydride, which was good with metal oxide catalysts.
ZnO was more effective as a catalyst than a coupling agent, enhancing the formation of cyclic dimers, the first stage in the polymerization of PLLA (ring-opening), than the second step (ring-opening).

Thermal gravimetric analysis (TGA)
TGA of the synthesized PLLA The synthetic PLA's thermal tolerance was observed y the loss in sample weight during heating at a programmed rate of 10 °C/min under a N 2 gas stream.It is most likely that the loss of ester groups causes PLLA to degrade at temperatures between 300 and 400 °C38 .According to the circumstances in Table 11, the TGA created by PLLA demonstrated reasonable thermal stability.
When PLLA was made using ZnO as a catalyst, the only actual decomposition began at 144 °C and reached its peak rapid weight loss of about 100% at 290 °C, but this occurred at 298 °C when ZnO was replaced with ZnCl 2 .When PLLA is synthesized with Sb 2 O 3 or SnCl 2 . 2 H 2 O, 100% of the weight is lost at 340 °C, but when Fe 2 O 3 is used as the catalyst, both the thermal stability and the weight loss are maximized at 311 °C.Maximum www.nature.com/scientificreports/thermal decomposition of PLLA occurred at 277 °C when ZnO was used as a catalyst and p-Toluene sulphonic acid were used as the coupling agent, but maximum thermal degradation occurred at 348 °C when MgO was used as the catalyst.
ZnO was used as a catalyst, and ZnCl 2 as a coupling agent in the synthesis of PLLA, which produced 100% breakdown at 276 °C.

TGA of encapsulated isosorbide dinitrate in PLLA nanoparticles
Figure 3a, b depicts TGA of PLLA samples synthesized by ZnO, ZnCl 2 , ZnO/ZnCl 2 , ZnO-p-toluene sulphonic acid (2:2), (2:1), TGA of PLLA samples synthesized by Fe 2 O 3 , Sb 2 O 3 , SnCl 2 , and MgO/p-Toluene sulphonic acid (2:1).Also, TGA was used to investigate the thermal stability of encapsulated isosorbide dinitrate nanoparticles (Fig. 3c).According to the findings; nanoparticles are more thermally stable than individual PLLA.Additionally, it is easy to notice that isosorbide encapsulation significantly increases thermal stability (Table 12).And by adding more isosorbide dinitrate, the thermal stability was improved.DSC of the synthesized PLLA As can be seen from the DSC thermogram (Fig. 4), PLLA exhibited a transition temperature of 47 °C, with a corresponding melting temperature of 130 °C.In contrast, the endothermic peak of pure isosorbide dinitrate manifested itself at its crystalline melting point of 126.6 °C.The significant decrease in the melting temperature of isosorbide dinitrate in the nanoparticles (Fig. 5) indicates that it likely transforms from a crystalline to an amorphous phase during the manufacturing process or dispersion in the PLLA matrix.Detailed data on these temperature fluctuations can be found in Tables 13 and 14.

X-ray diffraction analysis
On various regions of every sample, X-ray diffraction was carried out.The XRD scans of the produced PLLA in the range of 2Ɵ = 5-80° were displayed in Fig. 6a, b.The primary diffraction peaks for PLLA at 2Ɵ = 16.40° were assigned to the orthorhombic (2-0-0) or octahedral (1-1-0) crystal planes, demonstrating that the PLA crystal had this characteristic orthorhombic structure.At 2Ɵ = 18.60°, a second, less intense peak that matched the crystal plane appeared (2-0-3).Additionally, this suggests that the crystal structure is orthorhombic.Other people also reported a similar outcome 39 .The peaks of PLLA were displaced to 2Ɵ = 17.3° and 20.2° by loading the drug, indicating that the encapsulation of the drug led to an increase in the crystallinity of PLLA, according to the XRD pattern of isosorbide dinitrate and encapsulated nanoparticles in Fig. 6c.

Particle size and zeta potential analysis
Dynamic light scattering (DLS) was used to determine the particle size diameters of the nanoparticles encapsulating isosorbide dinitrate in the PLLA matrix, as well as their polydispersity index (PDI) and zeta potential, confirming their size and stability (Table 15).The observed particle size, ranging from 213 to 452 nm, is influenced by factors such as the type of surfactant, the polymer-to-drug ratio and the molecular weight of PLLA.A polydispersity index in the range of 0.38 to 1.00 indicates robust stability in water, while the zeta potential in the range of − 8.8 to − 22.4 supports the stability of the prepared particles in an aqueous environment.The negative zeta potentials are attributed to the presence of unbound carboxylic acid end groups on PLLA 40 .
During emulsification, a substance that reduces surface tension is used to stabilize the droplets.Oil-in-water emulsions can be stabilized with various surfactants, including ionic and non-ionic polymer surfactants.Careful selection of surfactants is crucial to achieve particle sizes in the nano range.In addition to stabilizing the emulsions during the nano emulsion process, the type and concentration of surfactant play a crucial role in preventing particle aggregation and maintaining a low polydispersity index.In the context of pharmaceutical applications, toxicity and biocompatibility considerations of surfactants are essential.

Effect of type of surfactant on particle size
PDI values and mean particle sizes for four samples made with various surfactants, including (Pluronic F88, Tetronic 1307, Tween 80, and PVA).In this study, it was discovered that Tetronic 1307, a surfactant, produces smaller nanoparticles than Pluronic F88, suggesting that it may be able to prevent the coalescence of oil globules.Tetronic molecules tend to align near the droplet's surface, reducing the free energy at the interface between the two phases and preventing the droplets from coalescing.The emulsion generated with this drug was unstable, and phase separation occurred after a few hours of emulsification, producing polymer aggregates.Tween 80 and polyvinyl alcohol were found to be insufficient as surfactants.

Effect of the ratio between polymer and drug on particle size
It was discovered that the ratio of the polymer to the drug impacted particle size.While maintaining the same organic phase volume, increasing the polymer content causes the organic phase's viscosity, which increases the viscous forces preventing droplet breakup and causing larger oil droplets to form, increasing particle size.Previous reports with comparable outcomes can be found in the literature 41 .

Effect of molecular weight of polymer on particle size
The relationship between the polymeric solution's viscosity and molecular weight is straightforward.Similar experimental settings showed that a low-viscosity polymer solution could disperse in an external aqueous www.nature.com/scientificreports/medium with small globules more effectively than a high-viscosity polymer solution, decreasing the size as the molecular weight increased 42 .The particle size was 291 nm for PLLA with a molecular weight of 24,165 Dalton but 213 nm for PLLA with a molecular weight of 11,051 Dalton.

Scanning electron microscope of encapsulated nanoparticles (SEM)
The morphological analysis of the nanoparticles encapsulating isosorbide dinitrate was carried out using SEM, as shown in Fig. 7.The SEM images clearly show the existence of aggregated rod-like particles tightly packed in the sample.The observed aggregation of these rod-like structures is likely the result of interactions and integration of reactants with various surfactant aggregates 43 .Furthermore, using Zetasizer measurements, we found that the particle size ranges from 213 to 452 nm, providing valuable insights into the size distribution of the encapsulated nanoparticles.This comprehensive morphological and dimensional characterization improves the understanding of the nanostructure and enables a more sophisticated interpretation of the encapsulation process and its potential effects.In vitro release of encapsulated isosorbide dinitrate in PLLA nanoparticles and kinetics of release In vitro release data, monitored by UV as mentioned in the experimental section, were used to better understand the mechanism of isosorbide dinitrate release from PLLA nanoparticles.The E.E% and D.L% for the different formulas are shown in Table 15.For F9, the E.E% and D.L% were the highest among all formulas reaching 84.1 and 26.8% as Pluronic F88 was used for the preparation of samples F8 and F9.Pluronic-based drug delivery systems (DDS) are known for their remarkable ability to enhance the solubility of drugs.This property not only protects the drugs from potential hazards such as hydrolysis, enzymatic degradation or unwanted conjugation with biomolecules, but also helps to preserve their therapeutic efficacy.These DDS serve different purposes.Some focus on prolonged drug release, while others are designed for targeted delivery.For the latter, the aim is to exploit additional cellular release mechanisms or to ensure intracellular release in order to optimize the therapeutic effect.When developing an ideal nanoformulation, the main objective is to ensure that the drug is retained for the required period of time and released precisely at the site of action, while maintaining therapeutic concentrations for maximum efficacy.The generated data were examined using various kinetic models previously described in the literature 44 .The release from the system is represented by the zero-order kinetic model, in which the release rate is independent of the concentration of the dissolved drug.Q 0 represents the initial dose, Q t represents the cumulative amount of medication released at a time "t, " and K 0 represents the zero-order release constant.
The identical approach was previously used 45 .
Where the release rate is concentration dependent is defined by the first-order release.
where K 1 is the first-order release constant.Higuchi defined the release of drugs from an insoluble matrix as a square root of a time-dependent process based on the Fickian diffusion: where KH is the Higuchi constant.
The Hixson-Crowell cube root law defines the release from systems by dissolution where there is a change in the surface area and diameter of particles.
where K HC is the Hixson-Crowell constant.
Korsmeyer et al. derived a simple relationship, the Korsmeyer-Peppas model, which described drug release from a polymeric system.K KP is the Korsmeyer-Peppas constant, and n is the release exponent describing the drug release mechanism 41 .The parameters that offer the best fit between experimental findings and the nonlinear function were used in the modeling process.The model that best fits the release date was chosen based on the correlation coefficient (R 2 ) in the models mentioned above.The model with the highest R 2 value best fits the release data.
The data show a two-stage release profile of isosorbide dinitrate (Fig. 8).During the first three hours of release, the isosorbide dinitrate bound to the surfaces of the nanoparticles was released.After 24 h, the amount of isosorbide dinitrate started to increase again, indicating that the drug contained in the PLLA NPs had started to diffuse.Since the carboxyl groups were less degraded in an acidic medium than in an alkaline medium, the effect of pH on the release behavior in fast formulas showed that the release was slower at lower pH values.This could be due to the fact that ionic interactions are relatively negligible in an acidic medium.The results of applying mathematical models to the release data (Table 16) showed that the Korsmeyer-Peppas semi-empirical model with n 0.3 provided an ideal fit.The goal of sustained release of isosorbide dinitrate from PLLA NPs was to improve patient compliance by achieving prolonged therapeutic drug concentration at the target site, reducing doses used, limiting side effects, reducing dosing frequency, modifying and improving isosorbide efficiency.

Cytotoxicity of encapsulated isosorbide dinitrate nanoparticles
To ensure that isosorbide dinitrate is not hazardous to normal cells, the viability of the cells was assessed using the SRB assay on the (HSF) cell line.Figure 9 shows the morphology of the nanoparticle-treated cells to show that the nanoparticles did not impact the morphology of the cells.For each formula, the IC 50 values were greater than 100, indicating the safety of the PLLA nanoparticles loaded with isosorbide dinitrate.Also, Fig. 10 shown Dose-response curve of F1, dose-response curve of F2, and dose-response curve of F5. ( 6)

Conclusion
The direct polycondensation method was used to synthesize PLLA.The FT-IR spectra showed no chemical interaction between the drug and polymer throughout nano encapsulation.The results demonstrated that PLLA yielded a greater M.wt using a solvent mixture than with xylene alone.The highest M.wt (25,114 Dalton) was obtained using antimony trioxide as a catalyst.The degree of polymerization increased as the catalyst concentration increased.Maleic anhydride was an effective coupling agent with ZnO in the polymerization of PLLA.P-toluene sulphonic acid was a more fabulous coupling agent with metal chloride catalysts than maleic anhydride, which was decent with metal oxide catalysts.XRD indicated that the encapsulation of the drug led to an increase in the crystallinity of PLLA.The results revealed that the release of the drug became slower in lower pHs, and the Korsmeyer-Peppas semi-empirical model with n 0.3 provided the greatest fit.The cytotoxicity of encapsulated isosorbide dinitrate nanoparticles showed that the IC 50 values were greater than 100, representing the safety of the PLLA nanoparticles loaded with isosorbide dinitrate.

Figure 2 .
Figure 2. (a) Effect of the ratio between MgO/p-TSA and MgO/MA, (b) Effect of the ratio between ZnO and (p-TSA, MA, and PA), (c) effect of coupling agents with ZnCl 2 , and (d) Effect of change between catalyst and coupling agent in M.wt of PLLA.

Table 2 .
Effect of the ratio between ZnO and different coupling agents.

Table 3 .
Effect of type of coupling agents with ZnCl 2 .

Table 4 .
Effect of change between catalyst and coupling agent.

Table 5 .
The effect of surfactant types.

Table 6 .
The effect of the ratio between PLLA and isosorbide nitrate.

Table 7 .
The effect of molecular weight of PLLA.

Table 8 .
1HNMR peaks in each sample of prepared PLLA.

Table 9 .
13CNMR peaks in each sample of prepared PLLA.Effect of type of catalystDifferent metal oxides and metal chlorides were used as catalysts to study the impact of catalyst type on the polymerization of PLLA.The M.wt 25,114 Dalton yield of antimony trioxide was the highest, according to the results.Table10summarizes these data.

Table 11 .
Thermal stability data of PLLA samples.

Table 12 .
Thermal stability data of encapsulated isosorbide dinitrate.

Table 13 .
DSC data of PLLA samples.

Table 15 .
The particle size by number and volume, PDI, zeta potential, E.E, and D.L of prepared Nanoparticles of isosorbide dinitrate encapsulated into PLLA.